The ability to measure trace quantities of biological materials in a variety of matrices is important to numerous fields. For example, the measurement of a few Bacillus anthracis spores in air would be invaluable to homeland security, the measurement Escherichia coli in water or Salmonella enterica in food would be invaluable to public safety, and the ability to measure disease causing bacteria (e.g. methicillin resistant Staphylococcus aureus, MRSA), disease biomarkers or nucleotides in body fluids, such as blood, saliva, or urine, would be invaluable to medical diagnosis. Three methods are widely used to detect such trace quantities of biological material, 1) culture growth of bacteria or viruses that are detected by eye with or without the aid of staining and a microscope, 2) immunoassays in which the binding of an antigen to an antibody is detected or, more recently, 3) polymerase chain reactions in which primers are used to separate target nucleotides, and polymerases are used to generate millions of copies of the nucleotides until they are detectable. The latter two methods often employ fluorescent or radioactive labels for detection.
For most of the applications described above, effective analysis requires speed, sensitivity, selectivity and, ideally, ease-of-use and portability to make at-site measurements. Rapid detection of B. anthracis spores in air is required for effective evacuation of potentially exposed personnel; rapid detection of a patient infected with MRSA is required to quarantine the patient and minimize spread of this disease; and rapid detection of E. coli in food is required to limit distribution of spoiled food that may cause widespread illness. Extreme sensitivity is also required, as the Department of Defense has estimated that the median lethal dose (LD50) for weapons-grade inhalation anthrax is as few as 2,500 B. anthracis spores (Defense Intelligence Agency, Soviet biological warfare threat, DST-1610E-057-86, 1986), while 106 colony forming units (CFUs) of MRSA can cause an infection (Freitas, R., “Microbivores: Artificial Mechanical Phagocytes Using Digest and Discharge Protocol”, Journal of Evolution and Technology, 14, 55-106, 2005), and 100 CFUs of E. coli on a food product, considered an infectious dose, can become 1 billion CFUs during a period of just eight hours of transport to market (E. coli CFUs double about every 20 minutes, Irwin, P L et al., “Evidence for a bimodal distribution of Escherichia coli doubling times below a threshold initial cell concentration”, BMC Microbiology, 10, 207, 2010). Specificity is also important, in that bacteria within a genus, species, subspecies, strain or serotype, such as B. cereus and S. aureus (non-methicillin resistant), can give false positive responses for B. anthracis and MRSA, members of the same genus and species respectively, using some analytical methods, potentially causing unnecessary anguish or treatment.
Unfortunately, none of the three methods described above can meet the listed requirements: culture growth takes several days, PCR takes several hours, and immunoassays lack sensitivity. In addition, only immunoassays are sufficiently portable for at-site measurements, such as at a military base or a food-processing center.
Recently, several researchers have proposed the use of surface-enhanced Raman spectroscopy (SERS) as the detection method for immunoassays. SERS involves the absorption of incident laser photons within nanoscale metal structures, generating surface plasmons, which couple with nearby molecules (the analyte) and thereby enhance the efficiency of Raman scattering by six orders of magnitude or more (Jeanmaire DL, RP Van Duyne, “Surface Raman Spectroelectrochemistry”, J Electroanal Chem, 84, 1-20, 1977; or Weaver MJ, S Farquharson, M A Tadayyoni, “Surface-enhancement factors for Raman scattering at silver electrodes: Role of adsorbate-surface interactions and electrode structure”, J Chem Phys, 82, 4867-4874, 1985).
SERS has been shown to be capable of detecting trace quantities of dipicolinic acid, a chemical prevalent in spores, such as B. anthracis, rapidly, and in an easy-to-use format (Farquharson et al., U.S. Pat. No. 7,713,914). However, specificity was limited, and bacilli could not be differentiated from clostridia; and differentiation at the species level (e.g. B. cereus versus B. anthracis) was not even a consideration. In light of immunoassays, it is reasonable to consider the use of a binding agent, such as an antibody, to selectively bind a target analyte, such as an antigen to achieve species, subspecies, strain or serotype selectivity. The concept was first demonstrated by coating a roughened silver electrode with anti-human thyroid stimulating hormone antibody (anti-TSH) to capture the TSH antigen (Tarcha et al U.S. Pat. No. 5,266,498; 1993). However, “no” SERS of this binding event was shown. Antibody capture was only demonstrated when a second anti-TSH antibody functionalized with a dye (2-[4′-hydroxyphenylazo]-benzoic acid) was added, which itself attached to the bound TSH antibody to produce a spectrum of the dye. Furthermore, the laser wavelength was matched to the absorption of the dye to generate resonance Raman scattering, which, like SERS, is known to enhance Raman scattering by as much as six orders of magnitude. Yet this combined surface-enhanced Raman and resonance Raman spectroscopic (SER(R)S) approach only achieved a modestly low concentration detection of 4 microg/mL (4×106 International Units/mL) for the TSH antigen when 40 microg/mL (10 times the concentration) of the dye-anti-TSH complex was added. This result is questionable, since the addition of 40 microg/mL of the dye-anti-TSH complex to a sample containing “no” TSH antigen produced a more intense dye spectrum, suggesting that the spectrum was due more to the dye being in direct contact with the metal surface as opposed through binding to the TSH antigen in forming a dye-(anti-TSH)-TSH-(anti-TSH)-silver complex. In any case, the authors stated that no spectra of antibodies or antigens were obtained, but only spectra of a dye, and antigen-antibody binding typically required an hour or more (two hours for the above example).
In the 19 years since the Tarcha patent there have been only a few publications in which a biochemical of interest, such as those described above, has been successfully bound to an antibody attached to a SERS substrate and detected by SERS. This is due to the fact that the target analyte must be within the plasmon field of the surface-enhanced Raman active (SER-active) metal to achieve a significant enhancement of the Raman signal, since the enhancement decreases with distance to the 12th power. This means that the plasmon field extends 10 nm at most. The challenge, and lack of sensitivity, has been elegantly demonstrated for the binding of E. coli to an antibody coated on silver nanoparticles (Naja et al., “Raman-based detection of bacteria using silver nanoparticles conjugated with antibodies”, Analyst, 132, 679 (2007). It was found that the signal was enhanced only by a factor of 20, far less than the expected 1 million or so, even when a very small antibody, Protein A, brought the bacteria to a distance of 8 nm from the metal surface. Furthermore, the E. coli sample was allowed to bind (incubate) to the antibody functionalized silver particles “overnight”, and dried on a glass slide to concentrate the sample prior to SERS measurements. Clearly, the basic concept of using SERS as the detector for immunoassays does not provide sufficient sensitivity or speed for the applications discussed above.
To overcome the sensitivity limitation, a number of researchers have followed the approach of Tarcha et al, by adding dye molecules to the immunoassay. White et al. (U.S. Pat. No. 6,750,065) describe the use of antibodies that bind drug-dye complexes, such that the introduction of the drug will displace the drug-dye complex, which can be detected “downstream” again using a combined surface-enhanced Raman and resonance Raman spectroscopy approach. The patent details methods to synthesize three drug-dye complexes, but provides no information regarding the antibody nor data showing displacement or the drug-dye complex or the patented principle. Sun et al. (U.S. Pat. No. 7,485,471) describe the use of a dye-labeled antibody that binds a specific antigen, which in turn binds a second antibody that contains a seed particle that, in turn, is used to grow a SER-active particle, which will ideally interact with the dye. No data are supplied, such as the time required to grow the SER-active particle or how the dye will come to interact with it. Consequently, neither of these patents addresses the rapid decrease in signals due to distance from the metal nor the long binding times. In fact the value of using a dye label to increase sensitivity has been questioned by Zhang et al. (“Protein adsorption drastically reduces surface-enhanced Raman signal of dye molecules”, J Raman Spectrosc, 41, 952, 2010), who have shown that the SER signal intensity of dye-protein complexes added to silver colloids is “reduced” by several orders of magnitude compared to the dye by itself. They showed that a flourescein dye attached to either bovine serum albumin, lysozyme, trypsin, or concanavalin A produced “no” SER signal. Furthermore, they suggest that the signals observed for the other dyes used in their experiments may have resulted from the dyes coming into direct contact with the silver colloids, as the proteins were not bound to the silver colloids before introduction of the dye.
Based on the foregoing, it is believed that one of ordinary skill in the art would not expect a significant SER signal to be generated from an antibody-antigen pair bound to SER-active particles (especially when the antigens are micron-sized bacterial cells), even with the introduction of additional SERS particles after binding has been achieved; nor would the signal strength be expected to be sufficient to detect as few as 10 bacterial spores or CFUs; nor would it be expected that detection could occur in less than 20 minutes, i.e. without a long incubation time.